WO2005007233A2 - Application de stimulation electrique pour le genie tissulaire fonctionnel in vitro et in vivo - Google Patents

Application de stimulation electrique pour le genie tissulaire fonctionnel in vitro et in vivo Download PDF

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Publication number
WO2005007233A2
WO2005007233A2 PCT/US2004/019731 US2004019731W WO2005007233A2 WO 2005007233 A2 WO2005007233 A2 WO 2005007233A2 US 2004019731 W US2004019731 W US 2004019731W WO 2005007233 A2 WO2005007233 A2 WO 2005007233A2
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cells
construct
tissue
cell
cardiac
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PCT/US2004/019731
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WO2005007233A3 (fr
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Milica Radisic
Hyoungshin Park
Robert Langer
Lisa E. Freed
Gordana Vunjac-Novacovic
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Massachusetts Institute Of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation

Definitions

  • the method includes contacting a substrate with mammalian cells, thereby forming a cell-seeded construct, and cultivating the cell- seeded construct in perfusion in the presence of a biomimetic electrical stimulation for a time period to form a three-dimensional cell structure exhibiting structural and functional characteristics of a native tissue.
  • the method includes contacting a substrate having a dry thickness of at least 1.5 mm with mammalian cells, thereby forming a cell-seeded construct, and cultivating the cell- seeded construct in the presence of a biomimetic electrical stimulation for a time period to form a three-dimensional cell structure exhibiting structural and functional characteristics of a native tissue.
  • Cultivating the cell-seeded construct may include implanting the cell-seeded construct in vivo and placing the cell-seeded construct in electronic stimulation with a source of a biomimetic electrical stimulation.
  • the substrate used in the preparative methods is biocompatible. This is particularly important when the three-dimensional tissue- engineered construct produced is to be implanted in a patient.
  • the substrate may be biodegradable or non- biodegradable; it may comprise a naturally-occurring polymer, a synthetic polymer, or any combination of naturally-occurring and/or synthetic polymers.
  • the mammalian cells used in the preparative methods of the invention comprise cells of one cell type.
  • inventive methods of treatment are particularly useful when the tissue to be replaced or supplemented is one that is subject to electrical stimulation in vivo.
  • inventive methods may, for example, be applied for treating an individual suffering from tissue deficiency, damage or loss associated with a congenital heart disease or with an acquired heart disease.
  • the invention provides methods for testing three- dimensional tissue-engineered constructs in vitro. These methods comprise producing a construct according to the preparative methods described above, exposing the construct to a test factor, and observing a response of the construct to the test factor.
  • integration refers to a direct functional and structural connection between native host tissue and implanted engineered construct(s).
  • histocompatibility refers to the degree of similarity between the histocompatibility antigens of two individuals. Histocompatibility determines whether an organ transplant or engineered construct implant will be tolerated or rejected by the recipient's body.
  • the term “hemocompatibility” refers to the ability of any material, mechanical device or tissue-engineered construct to be in contact with blood without interacting with any blood components so as to cause their inappropriate activation or destruction. Hemocompatible medical devices or materials do not trigger adverse reactions such as platelet attachment, platelet activation, and complement activation that eventually lead to fibrin production and clot formation.
  • cell trans-differentiation refers to the process by which a cell changes from one state of differentiation to another.
  • stem celF refers to a relatively undifferentiated cell that has the capacity for sustained self-renewal, often throughout the lifetime of an animal or human, as well as the potential to give rise to differentiated progeny (i.e., to different types of specialized cells).
  • An "embryonic stem DC is a stem cell derived from a group of cells called the inner cell mass, which is part of the early (4 to 5 days old) embryo called the blastocyst. Once removed from the blastocyst, the cells of the inner cell mass can be cultured into embryonic stem cells.
  • embryonic stem cells can proliferate indefinitely, a property that is not shared by adult stem cells.
  • -An "adult stem celF is an undifferentiated cell found in a differentiated (specialized) tissue.
  • Adult stem cells are capable of making identical copies of themselves for the lifetime of the organism.
  • Adult stem cells usually divide to generate progenitor or precursor cells, which then differentiate or develop into "mature" cell types that have characteristic shapes and specialized functions.
  • Sources of adult stem cells include bone marrow, blood, the cornea and retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, the lining of the gastrointestinal tract, and pancreas.
  • Exemplary polysaccharides include starches, dextrans, celluloses, hyaluronic acid and its derivatives; exemplary proteins include collagen and gelatin.
  • Polysaccharides such as starches, dextrans, and celluloses may be unmodified or may be modified physically or chemically to affect one or more of their properties such as their characteristics in the hydrated state, their solubility, or their half-life in vivo.
  • the substrate includes a biocompatible, degradable polymer. Such polymers can be broken down by cellular action and/or by action of non-living body fluid components. A variety of biocompatible, degradable polymers are suitable for use in the preparation methods of the present invention.
  • Suitable mammalian cells for use with the inventive methods of preparation of three-dimensional tissue-engineered constructs are cells that have a native capacity for differentiation into a particular tissue or cells that may be manipulated into forming a particular tissue. Therefore, suitable mammalian cells include neonatal cells, autologous or heterologous adult or aged donor cells, progenitor or precursor cells, and stem cells as long as they can be manipulated to form a given tissue. In one embodiment, the mammalian cells are (or can become, for example, after differentiation) electrically excitable.
  • mesenchymal stem cells which reside within the bone marrow cavity, have been shown, both in culture and following injection into particular tissues in mammals, to give rise to a range of cell types including chrondrocytes, osteoblasts, adipocytes, cardiac and skeletal muscle cells (K.W. Liechty et al, Nat. Med. 2000, 6: 1282-1286; M.F. Pittenger et al, Science, 1999, 284: 143-147), as well as cells typical of the central nervous system, such as neurons and astrocytes (G.C. Kopen et al, Proc. Natl. Acad. Sci. USA, 1999, 96: 10711-10716; D. Woodbury et al, J.
  • mammalian cells that can be used in the preparation methods of the present invention include precursor cells. Usually, between the stem cell and its terminally differentiated progeny state, there is an intermediate population of committed progenitors with limited proliferative capacity and restricted differentiation potential. These cells, called progenitor or precursor cells, are sometimes known as transit amplifying cells.
  • scaffolds are 1.5 mm thick (dry thickness) or greater, for example, at least 2 mm, at least 3 mm, or at least 4 mm thick.
  • perfused bioreactor systems have been developed (R.L.
  • the in vitro cultivation of the cell-seeded construct is carried out under conditions selected to promote deposition of extracellular matrix components.
  • the in vitro cultivation of the cell-seeded construct may also be carried out under conditions that promote cell proliferation and/or cell differentiation.
  • the cultivation dish or bioreactor is placed into an incubator, which provides a controlled environment.
  • the temperature of the incubator is generally set at 37°C, and the relative humidity of the incubator is held at 90%).
  • the composition of the gas environment in the incubator depends on the selection of the medium. For example, a mixture of air with 5% CO 2 may be used in combination with a bicarbonate-buffered medium, while air alone may be used when a HEPES-buffered medium is utilized.
  • cardiac tissue constructs of the invention are preferably cultivated in the presence of an electrical stimulation that mimics the electrical stimulation received by a cardiac muscle tissue in vivo.
  • the electrical stimulation is used to differentiate cardiac progenitor cells and/or stem cells into cardiac myocytes.
  • results from experiments carried out by the applicants suggest that when cardiac-like electrical stimulation is applied during in vitro cultivation, there is no need for chemical factors (such as, for example, 5-azacytidine) to induce cell differentiation. Therefore, cardiac-like electrical stimulation of human mesenchymal stem cells can be used to generate cardiac-like cell phenotype without the application of any specific chemical factors.
  • the inventive methods lead to the formation of cardiac muscle constructs with improved structural and functional characteristics.
  • the methods of preparation of the invention may further include the step of stimulating the cell-seeding construct mechanically and/or chemically.
  • Eschenhagen and coworkers have submitted cardiac patches formed using cardiac muscle cells from newborn rats to stretching (C. Finlc et al, FASEB J. 2000, 14: 669-679), and have shown that when these engineered tissues are implanted into rats, they contracted up to four times more vigorously than unstretched tissues (T. Eschenghagen et al, Transplant Immunol. 2002, 9: 315-321).
  • Other examples of mechanical stimulation include strain, hydrostatic pressure, direct compression, "high-shear fluid environments", and "low-shear fluid environments”. Multi-directional mechanical stimulation may also be used.
  • the present invention provides methods for treating an individual suffering from tissue deficiency, damage or loss. These methods may include producing a three-dimensional tissue-engineered construct according to the methods of preparation described above, implanting the construct into the individual in need thereof, and submitting the implanted construct to a biomimetic electrical stimulation.
  • the inventive methods of treatment are particularly useful when the native tissue to be replaced or supplemented is one which contains electrically excitable cells and is subject to electrical stimulation in vivo. Examples of such tissues include, but are not limited to, cardiac muscle tissue, striated skeletal muscle tissue, smooth muscle tissue, bone, vasculature, and nerve tissue.
  • the electrical stimulation applied to the construct in vivo following implantation may have several functions.
  • infarcted tissue i.e., scar tissue that is unable to contract during systole
  • infarcted tissue i.e., scar tissue that is unable to contract during systole
  • ventricular remodeling usually happens weeks or years after myocardial infarction. It corresponds to a progressive enlargement of the ventricle with depression of ventricular function, and is believed to result from the high stress undergone by tissues surrounding the initial infarction zone (D.K. Bogen et al, Circulation Res. 1980, 47: 728-741; J.
  • the presence and phenotype of the cells can be assessed by immunohistochemistry or ELISA using specific antibody, or by RT-PCR analysis.
  • Other types of constructs such as bioartificial equivalents of striated skeletal muscle tissue, smooth muscle tissue, bone, vasculature, or nerve tissue produced by the preparative methods of the invention may also be used in a large range of clinical applications.
  • nerve injuries have a significant impact on the quality of life. In the United States, an estimated 235,000 individuals suffer from physically debilitating spinal cord injuries, with total associated costs of more than $350 billion. Peripheral nerve injuries, such as facial paralysis and nerve damage in limbs resulting from accidents, occur even more frequently than spinal cord injury.
  • Drugs that are targeted to a specific tissue in the body may be tested using a corresponding inventive tissue equivalent to determine their safety, efficacy and mechanism of action by studying their effects on different properties of the tissue construct.
  • the effects of modulators or potential modulators of contractile activity may be studied using tissue-engineered cardiac muscle tissue.
  • Drugs may be tested for their effects on tissue maintenance and/or repair. It may also be desirable to use bioartificial cardiac muscle tissue to verify the safety and arrhythmogenic potential of drugs that are not targeted to the cardiovascular system but may produce unwanted side effects.
  • homogenates After centrifugation for 10 minutes at 12,000 g at 4°C, the homogenates are stored at -80°C for further analysis. On the day of the analysis, homogenates are diluted (1 part sample to 2 parts buffer) in Laemni buffer (Bio-Rad) containing 5% mercaptoethanol and 2%> SDS and boiled for 5 minutes to denature proteins. Homogenates containing 20 ⁇ g of total protein each are separated on 12% Tris-glycine minigels (Bio-Rad) using kaleidoscope prestained standards (Bio-Rad) at a constant voltage of 100 V for 2 hours at room temperature.
  • Figure 4 A is a photograph of the protein bands, showing that the intensity of the protein bands from stimulated constructs is greater than that from non- stimulated constructs.
  • Gene Expression Total RNA from the constructs is extracted using Trizol reagent (GibcoBRL) and RNeasy ® mini kit (Qiagen) following manufacturer's instructions. Briefly, constructs in Trizol are shredded by steel-ball bead beater using 6 cycles of 25 ⁇ m for 10 seconds each. The supernatant is collected after centrifugation at 14,000 ⁇ m for 15 minutes. Total RNA is collected by RNA column chromatography using water as eluant. RNA concentration is determined by measuring the absorbance at 260 nm by UN spectrophotometry. RT-PCR.
  • All microelectrodes are made of insulated tungsten wire and have uninsulated tips with diameters of 50 ⁇ 6 ⁇ m (Microprobe).
  • Two electrodes for bipolar stimulation are positioned 200 ⁇ m apart and connected to a programmable cardiac stimulator (SEC-3102, Nihon Kohden).
  • Eight recording electrodes are positioned 500 ⁇ m apart in a linear array, 1.5 to 5 mm from the stimulating site. Exact distances between electrodes are measured using a microscope and NIH 1.60 image analysis software. Shielded cables connect recording electrodes to bioelectric amplifiers (AB.601G, Nihon Kohden).
  • Spontaneous beating if present, is recorded for 3-5 minutes. After 15 minutes, monophasic pacing pulses (1-ms duration) are applied at a rate of 60 beats/minutes, starting at a pacing voltage of 0.1 V, which is then increased in 0.1 V increments until the sample is captured (z ' .e., until each pacing impulse is followed by a recorded tissue response).
  • the corresponding pacing voltage defined as the excitation threshold, represents the lowest stimulus that produces a stable propagation (for at least 1 minute at a rate of 60 beats per minute) over the length of the recording array.
  • the Matrigel suspension is seeded onto a UltrafoamTM collagen sponge scaffold (6 x 8 x 1.5 mm), which was first hydrated in culture medium (DMEM) for 2 hours and blotted dry.
  • the cell-Matrigel mixture is loaded to the scaffold, at the initial cell density of 8 x 10 7 cells/mL scaffold volume (using 60 ⁇ L of cell-Matrigel suspension per scaffold).
  • the construct is placed into a 6-well plate (one construct per well) and transferred to an incubator (humidified 5% CO 2 , 37°C environment) for 20 minutes in order for the gel to harden.
  • Cardiac differentiation medium (DMEM, 10% FBS, 1% Pen/Strep, 1% HEPES) is then added (5 mL per well) and the construct is cultured on an orbital shaker at 25 ⁇ m for 3 days. On Day 4, the construct is transferred into the stimulation chamber (a culture chamber equipped with the stimulation electrodes, as described above for the cultivation of cardiac myocytes). The electrical stimulation is applied for 7-8 days at a frequency between 60 and 180 bpm (1-3 Hz) and an amplitude of 5 N. Cardiac differentiation is assessed on-line, without interrupting the cultivation, by measuring the contractile responses (contractile amplitude, frequency) as described above for cardiac myocytes).
  • DMEM 10% FBS, 1% Pen/Strep, 1% HEPES
  • constructs were paraffin embedded, bisected, cross sectioned (5 ⁇ m thick), and either stained with hematoxylin and eosin or immunostained as described in Example 2 with monoclonal antibodies for sarcomeric ⁇ -actin (diluted 1:500, Sigma), cardiac troponin I (diluted 1:150, Biodesign), or sarcomeric tropomyosin (diluted 1:100, Sigma).
  • sarcomeric ⁇ -actin diluted 1:500, Sigma
  • cardiac troponin I diluted 1:150, Biodesign
  • sarcomeric tropomyosin diluted 1:100, Sigma
  • E Excitation threshold
  • MCR maximum capture rate

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Abstract

L'invention concerne des nouveaux procédés de préparation in vitro d'équivalents de tissu bioartificiel et leur intégration améliorée après implantation in vivo. Ces procédés consistent à soumettre une construction tissulaire à une stimulation électrique biomimétique pendant la culture in vitro afin d'améliorer ses propriétés structurelles et fonctionnelles, et/ou in vivo, après son implantation, de manière à améliorer son intégration avec le tissu hôte et augmenter la fonctionnalité et la survie cellulaire. Les procédés de l'invention sont particulièrement utiles pour la production d'équivalents bioartificiels et/ou la réparation et le remplacement de tissus natifs contenant des cellules pouvant être excitées électriquement et soumises à une stimulation électrique in vivo, comme par exemple, le tissu du muscle cardiaque, le tissu des muscles squelettiques striés, le tissu des muscles lisses, l'os, le système vasculaire et le tissu nerveux.
PCT/US2004/019731 2003-06-20 2004-06-21 Application de stimulation electrique pour le genie tissulaire fonctionnel in vitro et in vivo WO2005007233A2 (fr)

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